Important cellular functions like proliferation, differentiation, gene expression, cytoskeletal organization or cell survival depend on extracellular signals which lead to the activation of intracellular signaling pathways followed by a specific response appropriate for the biological needs of the cellular system. Signaling involves a large number of different proteins, and specific protein-protein interactions are a key event in the transduction of extracellular signals to the inside of the cell. Many signaling molecules contain domains also called protein-protein interaction modules (e.g. SH2-, SH3-, PTB-, PDZ- or WW-domains) composed of approximately 40 to 160 amino acids which can be separated from the original protein without loss of binding function. These domains contain ligand-binding surfaces that specifically interact with short linear sequence motifs (3 to 10 amino acids) in the complementary binding partners by which downstream signaling is mediated. Tyrosine-phosphorylated proteins involved in signaling are, for example, recognized by SH2- or PTB-domains and the specificity of the interaction is determined by the amino acid composition of the core binding site in the binding partner (pYxxΨ for SH2- or NPxpY for PTB-domains where pY=phosphotyrosine, N=asparagine, P=proline, Ψ=hydrophobic amino acids and x stands for any or selected amino acids which are important for the interaction). In contrast, SH3- or WW-domains bind to proline-rich sequences with the general consensus sequence ΨpxΨP for SH3-domains and the specificity of the interaction is additionally dictated by N-terminal or C-terminal localized amino acids flanking the core sequence. Binding affinities of these protein-protein interactions are in the range of 10−8 to 10−5 M (1, 2).
Existing Labeling and Detection Techniques
Today, a major problem in the detection of protein-protein interactions is the specific and efficient labeling of the protein-protein interaction domains. Different experimental approaches can be used for the labeling of protein-protein interaction domains. Labeling or tagging can either be performed before or after the purification of the recombinant expressed protein. Labeling of protein-protein interaction domains by biotin or 125I after the step of protein purification has been described previously (3, 4). A major disadvantage of covalent coupling methods is the control and unpredictable outcome of the labeling reaction. Inappropriate or overextensive labeling results in the loss of function of the binding protein and increased background, while low levels of labeling result in weak signals.
Moreover, labeling efficiency will vary between protein-protein domains as the number of reactive groups for coupling differ from domain to domain making the standardization of labeling and the subsequent quantification of binding interactions problematic. As the labeling conditions have to be determined experimentally for each domain, covalent labeling of recombinant proteins is not appropriate for the analysis of signaling networks, especially when a large number of differently labeled protein-protein interaction domains is needed.
Protein-protein interactions can also be detected without previous labeling e.g. by antibodies directed against the protein-protein interaction domain. Luttrell et al. used anti-src antibodies for the detection of the interaction of the src-SH2 domain with cellular proteins in a Far-Western blot (5). For the present invention, this approach is not appropriate, as a large number of non-crossreacting highly specific antibodies are needed which are not necessarily available. In addition, quantification of the protein-protein interactions is difficult, as the different antibodies will vary in their binding affinities resulting in variable signal intensities.
Alternatively, protein-protein interaction can be detected by tags that are expressed as parts of the fusion protein and are used for purification or are inserted in the fusion protein in addition to the purification tag. Kaelin et al. inserted the phosphorylation site of protein kinase A allowing the subsequent 32P-labeling of fusion proteins (6). Recently, Zhao et al. presented a vector system in which ras is inserted between the N-terminal localized GST-purification tag and C-terminal SH3-domains (7). Incubation of [γ-32P]GTP with the fusion protein results in the labeling of ras by which protein-protein interactions are detected. These approaches allow the uniform labeling of protein-protein interaction domains by radioactivity with the major disadvantage that only one label is available, so differential labeling of different domains cannot be achieved. Moreover, in the method described by Zhao et al. dissociation of the label will occur as the binding of GTP is not covalent. This results in the labeling of other protein-protein interaction domains that are supposed to behave as unlabeled competitors when this labeling method is applied to the competitive assay outlined in the invented approach.
Application of GST-Antibodies for the Detection of GST-Fusion Proteins
Additionally to the labeling and detection methods described above, protein-protein interactions can be directly detected by the purification tag, by using antibodies directed against the purification tag. This method has been used by Tanaka et al. for the detection of protein interactions applying GST-fusion proteins (8). We tested this approach for the detection of single SH2-domain interactions with cellular proteins in a Far-Western blot (FIG. 2). Protein lysates of 3T3 fibroblasts and v-abl transformed 3T3 fibroblasts were used as a model system and investigated in parallel. Due to the uncontrolled kinase activity of v-abl, strong tyrosine phosphorylation occurred in the v-abl transformed 3T3 fibroblasts in contrast to the untransformed cells. The difference in the extent of tyrosine phosphorylation between the two cell lysates can be demonstrated with the commonly applied antibody 4G10 which recognizes a broad spectrum of tyrosine phosphorylated proteins (FIG. 2A). For the Far-Western blot analysis, whole cellular lysates were prepared from both cell lines, applied in equivalent amounts to SDS-PAGE and proteins were transferred to PVDF-membranes. Incubation of the membrane was performed with a recombinant expressed GST-abl-SH2 domain or GST alone as a control. GST fusion is widely used for the expression and purification of recombinant proteins and GST-fusion proteins have already been successfully applied for the study of protein-protein interactions. After binding and washing, protein-protein interactions were detected by chemiluminescense (ECL) with an anti-GST mouse antibody directed against the non-denatured form of GST followed by incubation with a horseradish-peroxidase (HRP) labeled goat anti-mouse antibody. As demonstrated in FIG. 2B, differences in the tyrosine phoshorylation pattern of 3T3 and v-abl transformed 3T3 cells are detectable, similar to the pattern obtained with the 4G10 phosphotyrosine-specific antibody. However, the high background that was observed in the GST control mades this approach very problematic for the quantification and precise identification of differences in signal transduction patterns. Comparable results with high levels of background were also observed when the biotinylated GST-abl-SH2-domain was used as a probe and detection was performed with streptavidin-HRP (FIG. 2C). Additionally, the usefulness of the currently-available labeling systems (e.g. anti-GST antibody) for molecular diagnostics is severely limited because the low signal-to-noise results produced by these systems make it impossible to detect specific signals in cells, with few exceptions (such as the N54 cells which contain very high levels of tyrosine phosphorylation).
The qualitative and quantitative characterization of complex signaling networks and the identification of major cell type specific signaling proteins is very important because it would enable understanding of physiological processes of signal transduction and alterations of signaling in a multitude of human diseases such as cancer, autoimmunological diseases and other disorders. Detailed insights in signaling networks and the identification of disease-associated differences in signaling may lead to new ways for the rational design and development of specific drugs. The pattern of binding interactions in a cell or tissue may also be used as a tool for molecular diagnostics, for example in classifying tumors.
In general, signal transduction pathways and specific protein interactions are investigated by classical methods like Western blot analysis, Far-Western blot or co-immunoprecipitation studies as well as application of protein expression libraries or the two-hybrid assay system. In most of these applications single protein-protein interactions are studied. Techniques allowing the analysis of signaling networks do not currently exist. New methods for the analysis and characterization of complex cellular signaling networks are needed.